The leader peptidase (signal peptidase I) gene, lepB, of Rhodobacter capsulatus has been cloned and sequenced. The amino acid sequence of the predicted protein exhibits similarity to other known bacterial leader peptidases. R. capsulatus belongs to the alpha-subdivision of purple bacteria and thus is a relative of mitochondria in eukaryotes. Like the yeast mitochondrial inner membrane proteases IMP1 and IMP2, the leader peptidase from Rhodobacter has only one membrane-spanning segment. Sequence comparison of the Rhodobacter Lep protein with IMP1 and IMP2 did not reveal a higher overall similarity than between other prokaryotic signal peptidases and the mitochondrial enzymes. Expression studies using lacZ fusions in combination with primer extension analysis provide evidence for a weak promoter located a short distance from the transcription start of the lepB gene. Failure to establish a Rhodobacter strain with a disrupted lepB gene indicates that this gene is essential.